This proposal is to investigate the putative role of the ubiquitous lipidic polymer, polyhydroxybutyrate, in the sorting and membrane assembly to OmpA, the major outer membrane protein of Escherichia coli. The localization of proteins to different cellular compartments is a central process in the construction of a cell. Outer membrane proteins of Gram- negative bacteria must avoid entrapment in the cytoplasmic membrane, and yet achieve recognition by the outer membrane and proper assembly within it. These processes, known as protein sorting and assembly, are not understood. Studies have indicated that, in addition to the information within the signal sequence, information required for the localization and assembly of an outer membrane protein exists in a short amino acid sequence within the mature polypeptide. Such a sequence has been identified in OmpA. Our preliminary studies have shown that short-chain poly-3-hydroxybutyrate is covalently bound to an amino acid residue within this sorting sequence. OmpA protein will be fragmented by chemical and enzymatic digestion, and peptides containing polyhydroxybutyrate will be identified with anti- polyhydroxybutyrate IgG. The selected peptides will be purified by reverse-phase liquid chromatography, and sequenced by matrix-assisted laser desorption ionization mass spectroscopy to identify the amino acid covalently bonded to polyhydroxybutyrate. Site-directed mutagenesis, followed by in vitro translation, will be employed to study the effects of substitutions or deletions of this and neighboring amino acids on the incorporation of polyhydroxybutyrate into OmpA. Mutants which produced polyhydroxybutyrate-deficient OmpA protein will be developed by transformation of selected constructs into ompA-strains. The cellular location and proper assembly of the in vivo produced mutant OmpA proteins will be investigated by cell fractionation and immuno-electron-microscopy, and by examination of characteristic properties of properly assembled OmpA-heat modifiability and sensitivity to proteases.